The importance of including the C-terminal domain of PTP1B1-400 to identify potential antidiabetic inhibitors

Abstract In the present work, we studied the inhibitory and kinetic implications of classical PTP1B inhibitors (chlorogenic acid, ursolic acid, suramin) using three enzyme constructs (hPTP1B1-285, hPTP1B1-321, and hPTP1B1-400). The results indicate that the unstructured region of PTP1B (300-400 amino acids) is very important both to obtain optimal inhibitory results and propose classical inhibition mechanisms (competitive or non-competitive) through kinetic studies. The IC50 calculated for ursolic acid and suramin using hPTP1B1-400 are around four and three times lower to the short form of the enzyme, the complete form of PTP1B, the one found in the cytosol (in vivo). On the other hand, we highlight the studies of enzymatic kinetics using the hPTP1B1-400 to know the type of enzymatic inhibition and to be able to direct docking studies, where the unstructured region of the enzyme can be one more option for binding compounds with inhibitory activity.


Introduction
In recent years, the updated classification of diabetes mellitus (DM) published by the American Diabetes Association (ADA) in 2021 identifies four types of diabetes. (1) Type 1 diabetes (due to autoimmune-cell destruction, usually leading to absolute insulin deficiency, including latent autoimmune diabetes of adulthood), (2) Type 2 diabetes (due to a progressive loss of adequate b-cell insulin secretion frequently on the background of insulin resistance), (3) Specific types of diabetes due to other causes, and (4) Gestational diabetes mellitus (diabetes diagnosed in the second or third trimester of pregnancy that was not overt diabetes prior to gestation) 1 . The most critical clinical forms are type 1 DM (T1DM) and type 2 DM (T2DM). The latter is the type of diabetes for which between 90 and 95% of diagnosed cases of diabetes are registered worldwide. DM is a metabolic disorder characterised by hyperglycaemia, glycosuria, and some types of ketonaemia. These pathologies cause various complications, such as retinopathies, neuropathies, and peripheral vascular insufficiencies. The dramatic increase in DM in recent years may be related to multiple factors, including obesity and a sedentary lifestyle in the population 2 .
Due to the multifactorial nature of T2DM and the variety of molecular targets of the metabolic pathways associated with its aetiology, no specific pharmacological molecular target suggests a definitive treatment for this disease. However, various pharmacological targets for its treatment have been identified, such as dipeptidyl peptidase-4 enzyme (DPP-4), free fatty acid receptor 1 (FFAR1), G protein coupled receptors (GPCRs), a-Glucosidase, peroxisome proliferator-activated receptor (PPAR), augmenter of liver regeneration (ALR), G-protein coupled receptor for glucagon (GCGr), sodium-glucose linked transporter (SGLT), Phosphoenolpyruvate carboxykinase (PEPCK), and PTP1B, among the most important 2 .

Protein tyrosine phosphatases (PTPs)
The phosphorylation/dephosphorylation of tyrosine residues is a mechanism for regulating the activity of proteins at the cellular level; it is essential for the normal functioning of signal transduction networks associated with the receptors of different hormones, cytokines and other bioactive molecules 3 . PTPs are divided into three subfamilies, classified as (1) Specific tyrosine phosphatases, (2) Dual-specific phosphatases, and (3) Low molecular weight phosphatases 4 . Several PTPs, such as protein tyrosine phosphatase a (PTPa), type receptor protein tyrosine phosphatase LAR (PTP-LAR), and PTP1B, have been implicated in the negative regulation of the insulin signalling pathway. The most convincing data implicate PTP1B as the most important regulator of this process 5 .

Protein tyrosine phosphatases 1B (PTP1B)
PTP1B is a monomeric cytosolic protein encoded by the PTPN1 gene (locus 20q13), whose amino acid sequence includes 435 residues ($50 kDa) distributed in three structural domains: a highly conserved N-terminal catalytic domain known as PTP domain (PTP1B 1-300 ), an intrinsically disordered regulatory domain also known as proline-rich domain (PTP1B 301-400 ), and a hydrophobic C-terminal signalling domain (PTP1B 401-435 ) that directs the protein to the endoplasmic reticulum (ER) 6 . Knowledge of the structural domains of PTP1B has led to remarkable insights into the inhibition strategies targeted to the enzyme. On the one hand, the catalytic domain (PTP1B 1-300 ) is the target site for developing competitive, bidentate and allosteric inhibitors. Finally, the regulatory domain (PTP1B 301-400 ) is suggested as a second target site of allosteric inhibition. This second allosteric site is critical for interacting with potential inhibitors and enhancing their inhibitory potency 7 ( Figure 1).
Since the PTP1B catalytic domain has only been resolved by Xray diffraction, the protein forms used in most enzymatic assays and computational studies are PTP1B 1-300 and PTP1B 1-321 8 . This is justified by the intracellular physiological loss of signalling residues 400-435 that directs the protein to the ER and by the intrinsically unstructured nature of the region that comprises residues 300-400 of the protein, which adopts an extended structure in solution 6,7 . However, PTP1B 1-400 is the one found in vivo.
This phosphatase negatively regulates the insulin signalling pathway, promoting the dephosphorylation of phosphotyrosines (Tyr) in the b-subunit of the insulin receptor and probably in the IRS-1/2 protein 5,9,10 . In addition, this phosphatase also participates in the regulation of the leptin-dependent signalling pathway, and it is proposed that it is an essential regulator of the differentiation process of brown adipose tissue 5,11 (Figure 2).
The insulin released from the pancreatic beta cell is transported into the cell, and it binds with its receptor producing the insulin-IR complex. Then, the intracellular part of the receptor is autophosphorylated, turning into an active kinase (sensitization). The abnormal signalling causes stimulation of PTP1B, which is involved in the desensitisation of the receptor by dephosphorylation. Therefore, inhibition of PTP1B has been considered as a potential drug target for the design of promising inhibitors. The effect of such inhibitors consists in delaying the receptor desensitisation, prolonging insulin effect for the treatment of diabetes II 12 .
Increased PTP1B activity in peripheral tissues appears essential for establishing insulin resistance, as suggested by numerous studies. This enzyme's activity was increased in the muscle tissue of non-diabetic obese individuals suffering from insulin resistance 13 . On the other hand, inhibition of PTP1B expression by siRNA or gene deletion in laboratory mice increased their insulin sensitivity and improved glucose tolerance. In addition, it made them resistant to hypercaloric diet obesity 10,14 . Equally, it was shown that the inhibition of PTP1B expression in mice with polygenic insulin resistance (due to dysfunction of the insulin receptor and the IRS protein) improved glucose tolerance and insulin sensitivity and substantially decreased the incidence of diabetes 15 .
The background shows that PTP1B is a crucial protein in glucose homeostasis and is involved in the molecular mechanism of insulin resistance, a relevant condition in the pathogenesis of T2DM. Therefore, PTP1B can be an important therapeutic target in T2DM and other metabolic disorders such as obesity and metabolic syndrome 5,10 . Therefore, in recent years, there has been significant interest in the identification of PTP1B inhibitors, many of which have been identified from different sources and with various structural properties 7, [16][17][18][19][20][21][22] . Additionally, current efforts include both in vitro and in silico experiments to study, design and discover new potent and selective inhibitors 23-26 . There are some essential aspects in the search for PTP1B inhibitors; the most important are the potency, the type of inhibitor, and their selectivity. The first two can be performed using the appropriate enzyme form (hPTP1B 1-400 ). Despite the fact that the last one is challenging due to the homology of the PTP domain among the PTPs family members, it can oppositely provide an opportunity to discover activators from related PTPs inhibitors 27 . However, selectivity can be achieved by performing comparatives activity assays with T-Cell Protein Tyrosine Phosphatase (TCPTP), which is the closest homologue of PTP1B. In this work, we study the enzymatic inhibition and the kinetic effect of three variants of hPTP1B (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ) with the inhibitors ursolic acid (UA), chlorogenic acid (CGA), suramin (SUR), sodium orthovanadate (SO), and Compound 5 b a derivative of 18b-glycyrrhetinic acid and docking studies to complement the experimental data of the enzyme-ligand complexes. The inhibitors used were chosen because some are classical inhibitors and are used as positive controls in many previous works and because they present different types of inhibition (competitive, mixed and acopetitive). The results indicate that the unstructured region of hPTP1B (300-400 aminoacid) is crucial to obtain reliable inhibition and kinetic parameters. On the other hand, kinetic studies are of great value in establishing the type of inhibition of bioactive compounds of PTP1B.

Protein expression and purification
After DNA of each hPTP1B gene construct was sequenced (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ), they were transformed into E. coli strain BL21 (DE3). Transformed E. coli cells were grown in LB media (pH 7.5) containing kanamycin (30 mg/mL) at 37 C for 6 h with continuous agitation (250 rpm). Once the cultures reached an A 600 of 0.6 (about 6 h), protein expression was induced with 1 mM IPTG at 37 C, 250 rpm for 6 h. After centrifuge (4500 rpm, 15 min, 4 C), the grown and IPTG-induced cultures and bacterial pellet were resuspended in Tris buffer (pH 6.8) and lysed by sonication (10 cycles, intervals of 30 s) in an ice-water bath with an ultrasonic processor (Cole-Parmer, Vernon Hills, IL, USA). The cell lysate was centrifuged (13,000 rpm, 15 min, 4 C), the supernatant was filtered with a PVDF membrane (pore size 0.45 mm) (Argos, Vernon Hills, IL, USA) and loaded onto a HisTrap HP immobilised metal affinity chromatography (IMAC) column (Cytiva, Marlborough, MA, USA). The column was equilibrated with 50 mM Tris, pH 6.8, with three column volumes. His-tagged protein was eluted with Tris 50 mM and 300 mM Imidazole with one column volume. PTP1B phosphatase activity of the collected fractions was confirmed by the pNPP activity assay (see below). Fractions containing hPTP1B were dialysed in 50 mM Tris, pH 6.8. The purity of the protein was followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% resolving gel.
PTP1B activity assay hPTP1B activity assays using para-nitrophenyl phosphate (pNPP) as substrate were performed to determine the intrinsic catalytic activity of all three hPTP1B constructs (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ) confirmed in the purification process, and the in vitro inhibitory effect of the tested compounds. Initial experiments were carried out to set up suitable conditions for the following enzyme activity assays. The optimum substrate and enzyme concentration were determined by testing different concentrations of each. pNPP activity assays were carried out in a final volume of 100 mL of Tris 50 mM, pH 6.8, containing purified hPTP1B 66 nM and pNPP 0.25 mM. Enzyme solutions containing pNPP and inhibitors were incubated at 37 C for 15 min. The hydrolysis product's absorbance, para-nitrophenol (pNP), was measured every 60 s for 30 min at 405 nm using a 96-well microplate absorbance reader (Accuris). Absorbance values were expressed in terms of molar product using a pNP extinction coefficient of 18,000 M À1 cm À129 . The experiments were performed in triplicate. The variation of the molar pNP was analysed as a function of time, and the reaction rate was calculated as a function of pNPP concentration.

hPTP1B inhibition assays
The inhibitory effect of ursolic acid (UA), chlorogenic acid (CGA), and suramin (SUR) on the three purified hPTP1B constructs (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ) were assessed at the previously established optimal conditions (final volume of 100 mL of Tris 50 mM, DTT 0.25 uM pH 6.8 at 37 C for 15 min, absorbance lecture at 405 nm) using hPTP1B nM. IC 50 assays were carried out in the assay buffer at fixed enzyme concentration (66 nM of each hPTP1B construct), pNPP 0.25 mM, and ten increasing concentrations of each inhibitor (diluted in 100% DMSO) as follows: UA in 5 mM increments (5 to 50 mM), CGA in 0.4 mM increments (0.4 to 4.0 mM), and SUR in 1.0 mM increments (1.0 to 10 mM). The negative control was prepared with the enzyme and substrate in the absence of an inhibitor. The experiments were performed in triplicate. The percentage of inhibition was plotted as a function of the inhibitor concentration. IC 50 was calculated by regression analysis using Equation (1) (OriginPro 2018 (64-bit) SR1).
where % PTP1B is the percentage of inhibition, A 100 is the maximum inhibition, i is the inhibitor concentration, IC 50 is the concentration required to inhibit the enzyme's activity by 50%, and s is the cooperative degree.

Enzyme kinetics of hPTP1B inhibition
In a similar way to the IC 50 , the assays to determine the enzyme kinetic parameters and inhibition mechanism were conducted in the same experimental conditions but varying concentrations of pNPP in 0.1 mM increments (0.1-0.5 mM), and using five increasing concentrations of each inhibitor according to previously determined IC 50 V max is the maximum velocity, x is the inhibitor concentration, and K m is the Michaelis constant.
V max is the maximum velocity, x is the substrate concentration, I is the inhibitor concentration, K m is the Michaelis constant, K i is the inhibition constant, and a¼V max without inhibitor/V max with inhibitor.

Docking
The structural model of the hPTP1B1-400 protein was obtained from AlphaFold Protein Structure Database developed by DeepMind and EMBL-EBI (https://alphafold.ebi.ac.uk/). The pdb file was downloaded from the following link: https://alphafold.ebi.ac. uk/entry/P18031 30 . This structure was subjected to a molecular dynamics simulation for 50 ns to find the most stable conformation of the unstructured region (301-400) 31 . The structures of the ligands were constructed and minimised using AVOGRADRO software 32 . AutoDockTools 1.5.4 was used to prepare the pdb files of hPTP1B 1-400 and compounds. For stereoisomeric compounds, the conformer that presents greater stability after minimisation was coupled, maintaining the torsion angles detected by AutoDockTools. Polar hydrogen atoms and the Kollman unitedatom partial charges were added to the protein structures. In contrast, Gasteiger-Marsili charges and rotatable groups were automatically assigned to the structures of the ligands. We use Autodock Vina to do the docking, covering the entire enzyme 33 . The grid box size was 76 Â 56 Â 60 Å in the x, y, and z dimensions and central coordinates of 42, 66, and 59 for x, y, z, respectively to the active site; 50 Â 50 Â 50 Å in the x, y, and z dimensions and central coordinates of 67, 80, and 79 for x, y, z, respectively to the allosteric site 1, and 76 Â 56 Â 60 Å in the x, y, and z dimensions and central coordinates of 75, 51, and 53 for x, y, z, respectively to the allosteric site 2 with exhaustiveness of 8. Visualising the best conformational states was achieved with PyMOL version 2.4.0 and Maestro version 5.3.156 34 .

Results and discussion
Recombinant hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B  hPTP1B is an attractive molecular target recently for developing and discovering bioactive antidiabetic and anticancer molecules. Inhibitory activity assays have been performed with different forms of the enzyme, where inhibitory activity has been reported and the latter may be underestimated depending on the hPTP1B segment used and the type of inhibition of the compound to be reported. We constructed three forms of hPTP1B (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ) to evaluate different types of inhibitors (competitive and non-competitive). hPTP1B 1-285 and hPTP1B 1-321 represent the short form of hPTP1B that includes the N-terminal catalytic domain. hPTP1B 1-400 constitutes the long form of the protein that includes the intrinsically disordered C-terminal regulatory domain. Figure 3, shows the chromatogram of the purification by affinity chromatography and its follow-up by electrophoresis. The purified constructs showed a yield of 32, 40, and 56 mg per litre of culture medium for hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 , respectively. The K m of the sutrate (pNPP) is 16.37 ± 1.55, 15.66 ± 1.68, and 2.03 ± 0.11 mM for the hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 , respectively.   hPTP1B constructs (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ) (66 nM) were incubated with ten increasing concentrations of each inhibitor at 37 C for 15 minutes. Assay solutions (hPTP1B constructs þ assay buffer) without inhibitors were used as a negative control. The absorbance was measured at 405 nm. Data are representative of three independent experiments.
hPTP1B inhibition assays Figure 4 shows the inhibition assays of classical inhibitors with the three different forms of hPTP1B. The IC 50 observed for CGA is similar in all three enzyme forms. However, for the UA and SUR, the IC 50 estimated using hPTP1B 1-400 is around four and three times lower concerning the shorter forms (PTP1B 1-285 and PTP1B 1-321 ), respectively (Table 1). These results indicate that the IC 50 varies depending on the length of the hPTP1B used to perform the activity assay (short or long). Therefore, using the enzyme in its complete form (including the unstructured part, hPTP1B 1-400 ), which is the one found in vivo, has positive repercussions in calculating this parameter.

Enzyme kinetics of hPTP1B inhibition
The kinetic parameters are fundamental since we can determine the affinity of an inhibitor for the enzyme and its possible mechanism of inhibition. With this information, we can conduct directed docking studies and obtain three-dimensional models of the enzyme-inhibitor complex. For example, Figure 5 and Table 3 show the results of kinetic studies of 5 inhibitors using hPTP1B 1-400 . For the CGA, the K i is in the order of mM (1.03 mM), and the type of inhibition is competitive. Previous studies have reported non-competitive inhibition using hPTP1B 1-301 and hPTP1B 1-321 39,47 ; this discrepancy may be due to the type of enzyme used for carrying out the studies. Based on our kinetic assay, the type of inhibition proposed for the UA was mixed in the order mM (K i ¼4.0 mM). However, a competitive and non-competitive type of inhibition has been previously reported using hPTP1B 1-321 45 and PTP1B 1-298 44 , respectively. For the SUR inhibitor, both previous studies 28,40,50 and ours are in harmony when proposing a competitive inhibition, despite using different types of PTP1B. In the case of SO, the calculated K i is in the nanomolar order (K i ¼89 nM), and the type of inhibition was uncompetitive an order of magnitude higher K i (0.38 mM) and a type of competitive inhibition have previously been reported for

Docking studies
Based on the information from the kinetic studies, we docked three inhibitors with hPTP1B 1-400 (CGA, SUR and UA); the results are shown in Figures 6 and 7 and Table 4. CGA and SUR were directed to the catalytic site, and UA to the classically reported allosteric site and to the unstructured region where possible inhibitor-binding sites have also been recently reported 51 . Therefore, we have called site 1 the previously reported allosteric site and site 2 the one found in the unstructured region ( Figure  6). The details at the molecular level of the inhibitors with PTP1B indicate hydrogen-bridge, hydrophobic, and ionic (negative and positive) interactions, among the most important ( Figure 7). The theoretical binding parameters (K i theoretical) are in the range of magnitude similar to the experimental values (Table 3). Two possible allosteric sites were studied for the UA, where the K i were 22.23 and 4.75 mM, respectively. For the SUR and CGA inhibitors, the K i theoretical were 23.98 and 747.12 mM, respectively. These studies are relevant to analyse specific interactions at the molecular level and propose modifications to the inhibitors to try to improve their inhibitory activity or specificity on hPTP1B.

Conclusions
This work highlights the importance of using hPTP1B 1-400 in inhibition and kinetic assays. The IC 50 of the compounds CGA, UA, and SUR estimate using the three constructions of the enzyme (hPTP1B 1-285 , hPTP1B 1-321 , and hPTP1B 1-400 ), indicates that this parameter using the complete enzyme is lower for the inhibitors AU and SUR. Therefore the IC 50 of previously reported inhibitors that used the short forms of PTP1B may be underestimated. The complete form of hPTP1B is the one that is found in vivo in the cytosolic space, and it seems that the unstructured region plays an important role in the modulation of the enzyme's activity. Another critical aspect of PTP1B inhibitors is knowing their inhibition    mechanism, which can be known indirectly using enzyme kinetics studies, which can additionally be used to guide docking studies or molecular dynamics simulation. Molecular modelling studies can help improve known PTP1B inhibitors by analysing interactions at the molecular level of enzyme-inhibitor complexes and proposing synthetic derivatives. Based on the results obtained in this work, we can highlight some relevant points, such as (1) The importance of using the hPTP1B 1-400 enzyme to get binding and kinetic parameters.
(2) Carry out kinetic studies of the inhibitors to describe their mechanism of action. (3) Conduct in silico studies based on experimental studies, and (4) Redesign non-competitive inhibitors with low K i (high potency) to favour selectivity and potency for PTP1B.